Compositions for preventing or treating obesity comprising Sapium japonicum extract as an active ingredient

Abstract

The present invention relates to a composition for the prevention, treatment, or improvement of obesity comprising an extract of *Sapium japonicum* as an active ingredient. Since the *Sapium japonicum* extract of the present invention possesses anti-obesity activity that inhibits the process of preadipocyte differentiation into adipocytes and intracellular lipid accumulation, a composition comprising the *Sapium japonicum* extract is provided as a novel composition for the prevention, treatment, or improvement of obesity.

Description

Technology Field

[0001] The present invention is a human-shaped tree ( Sapium japonicum The present invention relates to a composition for the prevention, treatment, or improvement of obesity comprising an extract as an active ingredient. Background Technology

[0002] Obesity generally refers to a condition characterized by an excessive amount of adipose tissue in the body. It is a phenomenon where excess energy is accumulated as body fat because the energy consumed through food fails to balance with the energy expended through physical activity. Obesity is known to be caused by a wide variety of factors, including genetic factors, environmental influences such as irregular eating habits, lack of exercise, or the Westernization of dietary habits, psychological effects such as depression, boredom, and excessive stress, and pathological factors such as changes in thyroid or adrenal cortical hormones. Recently, due to economic growth and changes in lifestyle, dietary habits have undergone significant transformation. As a result, there is a rising trend of overweight and obesity among busy modern individuals, who consume high-calorie diets such as fast food and engage in insufficient physical activity.

[0003] Furthermore, since obesity is caused by the hypertrophy of adipocytes, inhibiting adipocyte differentiation can be helpful in treating obesity. Previous anti-obesity studies related to the inhibition of adipocyte differentiation and lipid accumulation have been reported, and 3T3-L1 mouse fibroblasts are generally used in in vitro studies as a well-characterized model for the differentiation of adipocyte progenitor cells.

[0004] The severity of obesity is recognized more significantly due to the various complications it can cause than due to the dangers of obesity itself. When body fat accumulates abnormally over a long period due to energy imbalance, it leads to various metabolic and adult diseases such as diabetes, hyperlipidemia, heart disease, stroke, arteriosclerosis, and fatty liver disease. Furthermore, obesity can cause not only physical ailments but also mental health issues such as social isolation, alienation, lack of confidence, and depression. Consequently, this is emerging as a serious social problem in Korea as well as in the West, and the necessity of preventing and treating obesity is being recognized as extremely important.

[0005] Obesity can be treated through lifestyle improvements such as dietary therapy and regular exercise, as well as medications like appetite suppressants and fat absorption inhibitors. Since obesity is a chronic disease, drug treatment requires long-term use; currently, products approved for long-term use of more than three months in Korea include the appetite suppressant sibutramine and the lipase inhibitor orlistat. However, most of these obesity treatment drugs are psychotropic substances that act on the central nervous system to regulate appetite, which are accompanied by side effects such as headaches and vomiting, and raise concerns regarding abuse. Therefore, active research is being conducted to develop materials with high safety and excellent anti-obesity effects that can resolve the side effects of the aforementioned commercially available anti-obesity drugs. Prior art literature

[0006] Korean Published Patent Application No. 10-2020-0056367 (Published May 22, 2020) The problem to be solved

[0007] The objective of the present invention is to provide a composition comprising a Japanese zelkova tree extract as a new composition for the prevention, treatment, or improvement of obesity by confirming that the extract has anti-obesity activity that inhibits the process of preadipocyte differentiation into adipocytes and intracellular lipid accumulation, thereby providing a new formulation for the prevention, treatment, or improvement of obesity. means of solving the problem

[0008] The present invention provides a pharmaceutical composition for the prevention or treatment of obesity comprising an extract of the Japanese spindle tree as an active ingredient.

[0009] In addition, the present invention provides a health functional food composition for the prevention or improvement of obesity comprising an extract of the Japanese spindle tree as an active ingredient. Effects of the invention

[0010] According to the present invention, since the extract of the Japanese spindle tree of the present invention has anti-obesity activity that inhibits the process of preadipocytes differentiating into adipocytes and intracellular lipid accumulation, a composition containing the extract of the Japanese spindle tree can be provided as a new composition for the prevention, treatment, or improvement of obesity. Brief explanation of the drawing

[0011] Figure 1 shows the results of evaluating the cytotoxicity of the Japanese spindle tree extract. Figure 2 is a photograph of the results of an analysis using Oil Red O staining to evaluate the effect of Japanese zelkova extract on intracellular lipid accumulation. Figure 3 is a graph showing the results of Oil Red O staining analysis to evaluate the effect of Japanese zelkova extract on intracellular lipid accumulation. Figure 4 shows the results of analyzing the mRNA expression levels of transcription factors related to adipocyte differentiation and lipid accumulation to evaluate the effect of Japanese zelkova extract on adipocyte differentiation. Figure 5 shows the results of analyzing the protein expression levels of transcription factors related to adipocyte differentiation and lipid accumulation to evaluate the effect of Japanese zelkova extract on adipocyte differentiation. Specific details for implementing the invention

[0012] The terms used in this specification have been selected based on currently widely used general terms whenever possible, taking into account their functions in the present invention; however, these terms may vary depending on the intent of those skilled in the art, case law, the emergence of new technologies, etc. Additionally, in specific cases, terms have been arbitrarily selected by the applicant, and in such cases, their meanings will be described in detail in the relevant description of the invention. Therefore, the terms used in this invention should be defined not merely by their names, but based on their meanings and the overall content of the invention.

[0013] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as generally understood by those skilled in the art to which the present invention pertains. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and should not be interpreted in an ideal or overly formal sense unless explicitly defined in this application.

[0014] Numerical ranges include the values ​​defined in the above ranges. All maximum numerical limits given throughout this specification include all lower numerical limits as clearly written. All minimum numerical limits given throughout this specification include all higher numerical limits as clearly written. All numerical limits given throughout this specification will include all better numerical ranges within a wider numerical range, as clearly written.

[0015] The present invention will be described in more detail below.

[0016] The present invention provides a pharmaceutical composition for the prevention or treatment of obesity comprising an extract of the Japanese spindle tree as an active ingredient.

[0017] The above extract of the Japanese zelkova tree is the Japanese zelkova tree ( Sapium japonicum It may be a fruit extract of (Siebold & Zucc.) Pax & Hoffm.

[0018] The above-mentioned Japanese zelkova extract exhibits anti-obesity activity by inhibiting the differentiation of preadipocytes into adipocytes and suppressing intracellular lipid accumulation.

[0019] In addition, the above-mentioned Japanese zelkova extract exhibits anti-obesity activity by inhibiting the expression of intracellular PPARγ, C / EBPα, and adiponectin.

[0020] The pharmaceutical composition of the present invention may be manufactured in a unit dose form or contained in a multi-dose container by formulation using a pharmaceutically acceptable carrier according to a method that can be easily carried out by a person skilled in the art to which the invention belongs.

[0021] The above-mentioned pharmaceutically acceptable carriers are those commonly used in formulations and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above components, the pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc.

[0022] In the present invention, the content of the additive included in the pharmaceutical composition is not particularly limited and can be appropriately adjusted within the content range used in conventional formulations.

[0023] The above pharmaceutical composition may be formulated into one or more external forms selected from the group consisting of injectable formulations such as aqueous solutions, suspensions, and emulsions, pills, capsules, granules, tablets, creams, gels, patches, sprays, ointments, warning agents, lotions, liniments, pastes, and cataplasms.

[0024] The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier and a diluent for formulation. The pharmaceutically acceptable carrier and diluent include, but are not limited to, excipients such as starch, sugar, and mannitol; fillers and extenders such as calcium phosphate; cellulose derivatives such as carboxymethylcellulose and hydroxypropylcellulose; binders such as gelatin, alginates, and polyvinylpyrrolidone; lubricants such as talc, calcium stearate, hydrogenated castor oil, and polyethylene glycol; disintegrants such as povidone and crospovidone; and surfactants such as polysorbate, cetyl alcohol, and glycerol. The pharmaceutically acceptable carrier and diluent may be biologically and physiologically affinity to the target. Examples of diluents include, but are not limited to, saline solution, aqueous buffer solution, solvent, and / or dispersion media.

[0025] The pharmaceutical composition of the present invention may be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically) depending on the intended method. For oral administration, it may be formulated into tablets, troches, lozenges, water-soluble suspensions, oily suspensions, prepared powders, granules, emulsions, hard capsules, soft capsules, syrups, or elixirs. For parenteral administration, it may be formulated into injectable solutions, suppositories, powders for respiratory inhalation, aerosols for sprays, ointments, powders for topical application, oils, creams, etc.

[0026] The dosage of the pharmaceutical composition of the present invention may vary depending on the patient's condition and weight, age, gender, health status, dietary constitutional specificity, properties of the formulation, degree of disease, time of administration of the composition, method of administration, duration or interval of administration, excretion rate, and form of the drug, and may be appropriately selected by a person skilled in the art. For example, it may be in the range of about 0.1 to 10,000 mg / kg, but is not limited thereto, and may be administered once or several times a day.

[0027] The above pharmaceutical composition may be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically) depending on the intended method. The pharmaceutical effective amount and effective dosage of the pharmaceutical composition of the present invention may vary depending on the formulation method, method of administration, time of administration and / or route of administration, etc., and a person skilled in the art can easily determine and prescribe a dosage effective for the intended treatment. The pharmaceutical composition of the present invention may be administered once a day or divided into several doses.

[0028] In addition, the present invention provides a health functional food composition for the prevention or improvement of obesity comprising an extract of the Japanese spindle tree as an active ingredient.

[0029] The present invention can be generally used as a commonly used food.

[0030] The food composition of the present invention may be used as a health functional food. The term "health functional food" refers to a food manufactured and processed using raw materials or ingredients having functional properties useful to the human body in accordance with the Health Functional Foods Act, and the term "functionality" refers to consuming the food for the purpose of obtaining useful effects for health purposes, such as regulating nutrients or physiological actions on the structure and function of the human body.

[0031] The food composition of the present invention may include conventional food additives, and unless otherwise specified, suitability as a "food additive" is determined in accordance with the specifications and standards for the relevant item, in accordance with the general provisions and general test methods of the food additive code approved by the Ministry of Food and Drug Safety.

[0032] Examples of items listed in the above "Food Additives Codex" include chemically synthesized products such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamon acid; natural additives such as persimmon dye, licorice extract, crystalline cellulose, sorghum dye, and guar gum; and mixed preparations such as L-sodium glutamate preparations, alkaline noodle additives, preservative preparations, and tar dye preparations.

[0033] The food composition of the present invention can be manufactured and processed in the form of tablets, capsules, powder, granules, liquid, pills, etc.

[0034] For example, among health functional foods in capsule form, hard capsules can be manufactured by mixing and filling a conventional hard capsule with an additive such as an excipient, and soft capsules can be manufactured by mixing the composition according to the present invention with an additive such as an excipient and filling it into a capsule base such as gelatin. The soft capsules may contain plasticizers such as glycerin or sorbitol, coloring agents, preservatives, etc., as needed.

[0035] The definitions of terms regarding the above excipients, binders, disintegrants, lubricants, synergists, flavoring agents, etc., are those described in literature known in the art and include those with identical or similar functions. There are no special restrictions on the types of food mentioned above, and they include all health functional foods in the conventional sense.

[0036] In this invention, the term “prevention” refers to any act of suppressing or delaying a disease through the administration of a composition according to this invention. In this invention, the term “treatment” refers to any act of improving or beneficially altering the symptoms of a disease through the administration of a composition according to this invention. In this invention, “improvement” refers to any act of improving a poor condition of a disease by administering or ingesting a composition of this invention to an individual.

[0037] Hereinafter, experimental examples and embodiments will be described in detail to aid in understanding the present invention. However, the following experimental examples and embodiments are merely illustrative of the content of the present invention and the scope of the present invention is not limited to the following experimental examples and embodiments. The experimental examples and embodiments of the present invention are provided to more completely explain the present invention to those with average knowledge in the art.

[0038] <Experimental Example> Experimental Materials and Methods

[0039] The following experimental examples are intended to provide experimental examples that are commonly applied to each embodiment according to the present invention.

[0040] 1. Sample preparation

[0041] Manju tree Sapium japonicum The fruit extract of (Siebold & Zucc.) Pax & Hoffm. was obtained from the Korea Plan Extract Bank (KPEB; Jeollanam-do) (Distribution No.: KPM025-071). The above-mentioned Chinese quince ( Sapium japonicumThe fruit extract was prepared by extracting the fruit parts of the collected Japanese spindle tree with 100% methanol to produce an extract, and then processing it into a powder form through filtration and vacuum evaporation drying. The Japanese spindle tree fruit extract was dissolved in DMSO (Dimethyl sulfoxide) at a concentration of 50 mg / ml and used.

[0042] 2. Cell Culture

[0043] 3T3-L1 cells, a preadipocyte line, were obtained from ATCC (ATCC CL-173). 3T3-L1 cells were cultured in DMEM (Dulbecco's Modified Eagle Medium) containing 10% Bovine Calf Serum (BCS; Gibco) and 1% penicillin / streptomycin at 37°C under 5% CO2 conditions. When the cell confluency reached 80% in a 100π dish, the cells were subcultured into a 6-well plate and cultured for 2 days.

[0044] 3. Fat differentiation

[0045] After reaching a confluency of 100%, 3T3-L1 cells were subcultured and cultured for 2 days, after which differentiation into adipocytes began. To differentiate 3T3-L1 cells into adipocytes, the culture medium was replaced with DMEM (Dulbecco's Modified Eagle Medium) containing 10% FBS (Fetal bovine serum; Gibco) containing MDI. The composition of MDI was as follows: 1 µg / mL insulin, 0.25 mM dexamethasone, 0.5 mM IBMX (3-Isobutyl-1-methylxanthine), and 0.125 mM indomethacin. After 2 days of differentiation, to promote adipocyte maturation, the medium was replaced with DMEM containing 10% FBS (Fetal bovine serum; Gibco) and 1 µg / mL insulin, and differentiation was carried out for a total of 8 days.

[0046] 4. Cell viability analysis

[0047] To evaluate the cytotoxicity of the extract, CCK-8 (Cell Counting Kit-8; Dojindo Molecular Technologies, Kumamoto, Japan) was performed. 3T3-L1 cells were placed in 96-well plates at a rate of 3 x 10⁶ per well. 4 Cells were inoculated and cultured for 24 hours. Japanese spindle tree extract or DMSO (Dimethyl sulfoxide) was added to each well and cultured for 48 hours. Afterward, CCK solution was added to each well and reacted at 37°C for 1 hour, after which the absorbance was measured at 450 nm.

[0048] 5. Oil Red O staining

[0049] 3T3-L1 cells were seeded into a 6-well plate and differentiated according to the adipogenesis process described above. On day 8 of differentiation, adipocytes were washed with PBS (phosphate buffer saline) and fixed with 4% paraformaldehyde for 1 hour. Subsequently, they were stained with filter-purified 0.3% Oil Red O solution at room temperature for 30 minutes. After staining, the cells were washed three times with distilled water and observed under a microscope. The lipid accumulation was quantified by dissolving in isopropyl alcohol and measuring the absorbance at 450 nm.

[0050] 6. RT-PCR(Real-Time Reverse Transcription Polymerase Chain Reaction)

[0051] 3T3-L1 cells were seeded into 6-well plates and differentiated according to the adipocyte differentiation process described above. On day 8 of differentiation, adipocytes were washed with PBS (phosphate buffer saline), and total RNA was extracted from the 3T3-L1 cells using Trizol reagent. Subsequently, mRNA was quantified using a nanodrop, and a cDNA library was synthesized using the PrimeScript™ RT Reagent Kit (TaKaRa Bio, Kyoto, Japan). mRNA expression levels were analyzed using SYBR Green (TOYOBO, Japan) and cDNA analysis implemented by the iCycleriQ™ Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA). mRNA expression levels were quantified as multiples of the mRNA expression level by comparing the relative expression levels to the β-actin expression level. The primer sets used are shown in Table 1 below.

[0052] Gene name Accession No. Sequence Adipoq NM_009605 Forward 5'- ACCTACGACCAGTATCAGGAAAAG-3' Reverse 3'- ACTAAGCTGAAAGTGTGTCGACTG-5' C / ebpα NM_001287523 Forward 5'- TTACAACAGGCCAGGTTTCC-3' Reverse 3'- GGCTGGCGACATACAGATCA-5' Pparγ AB644275 Forward 5'- TTTTCAAGGGTGCCAGTTTC-3' Reverse 3'- AATCCTTGGCCCTCTGAGAT-5' β-actin EF095208 Forward 5'- GACAACGGCTCCGGCATGTGCAAAG-3' Reverse 3'- TTCACGGTTGGCCTTAGGGTTCAG-5'

[0053] 7. Western Blot

[0054] 3T3-L1 cells were seeded into a 6-well plate and differentiated according to the adipogenesis process described above. On day 8 of differentiation, 3T3-L1 cells were dissolved in RIPA buffer (Radioimmunoprecipitation assay buffer) containing a phosphatase inhibitor and a protease inhibitor. Subsequently, Sodium Dodecyl Sulfate loading buffer (SDS loading buffer) was added, and the mixture was heated at 100°C for 10 minutes. The total protein amount was quantified to 30 μg using the Bradford protein assay. The quantified protein was separated by electrophoresis on a 7.5% to 15% SDS-polyacrylamide gel. Subsequently, the samples were transferred to a nitrocellulose membrane and blocked with 5% skim milk at room temperature for 1 hour. Then, the primary antibody corresponding to each protein was added, and the mixture was incubated overnight at 4°C. Afterward, the samples were washed with 1X TBS-T buffer (Tris buffered saline-tween buffer) and reacted with an HRP-conjugated secondary antibody for 1 hour. The antibody-conjugated proteins were detected using electrochemiluminescence (ECL) and a Fusion Solo Detector. Each protein band was quantified as a relative expression level based on the expression level of β-actin using Image-J software. The sieves used are listed in Table 2 below.

[0055] Gene name Company Product No. IgG PPARγ SCBT sc-7273 M C / EBPα CST 2295S R IRS1 CST 2390S R p-IRS1 Invitrogen 2103503 R Akt CST 4691S R p-Akt CST 4060S R β-actin SCBT sc-47778 M β-tubulin Abcam ab179513 R

[0056] Example 1. Evaluation of cytotoxicity of Japanese spindle tree extract

[0057] To evaluate the safety of the Japanese zelkova extract, cytotoxicity against 3T3-L1 cells was analyzed. After performing a maturation process for 8 days to differentiate into adipocytes, the differentiated 3T3-L1 cells were placed in 96-well plates at a rate of 3 x 10⁶ per well. 4 Cells were inoculated and cultured for 24 hours. Differentiated 3T3-L1 cells were treated with Japanese spindle tree extract at concentrations of 0.25 μg / mL, 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 4 μg / mL, 8 μg / mL, 16 μg / mL, or 32 μg / mL, and wells treated with DMSO (Dimethyl sulfoxide) were used as a control.

[0058] As shown in Figure 1, it was found that the viability of 3T3-L1 cells was not affected even when treated with SJF extract at a concentration of up to 32 μg / mL. The above results demonstrate that the SJF extract of the present invention is a safe substance with no cytotoxicity.

[0059] Example 2. Evaluation of the effect of Japanese spindle tree extract on lipid accumulation

[0060] To evaluate the effect of Japanese zelkova extract on intracellular lipid accumulation during the differentiation of preadipocytes into adipocytes, Japanese zelkova extract was applied during the adipogenesis process, and the degree of lipid accumulation was analyzed using Oil Red O staining. Japanese zelkova extract was diluted and applied to a medium containing MDI during the differentiation process of 3T3-L1 preadipocytes.

[0061] As shown in Figures 2 and 3, compared to undifferentiated 3T3-L1 cells (NC), 3T3-L1 cells with completed adipogenesis (WC) showed intracellular lipid accumulation, causing the cells to be stained red. On the other hand, when treated with 4 μg / mL or 8 μg / mL of SJF extract, the degree of lipid accumulation was found to be significantly reduced in a concentration-dependent manner compared to 3T3-L1 cells with completed adipogenesis (WC). The above results demonstrate that SJF extract has an effect of inhibiting the differentiation of adipocytes and intracellular lipid accumulation.

[0062] Example 3. Evaluation of the effect of Japanese spindle tree extract on adipogenesis factors

[0063] To evaluate the effect of Japanese zelkova extract on adipocyte differentiation at the molecular level, 3T3-L1 cells were treated with Japanese zelkova extract during the process of differentiation into adipocytes, and the expression levels of transcription factors related to adipocyte differentiation and lipid accumulation were analyzed.

[0064] Transcription factors such as PPARγ and C / EBPα are adipocyte differentiation factors whose expression is regulated to increase during the differentiation process, and adiponectin, HSL (hormone-sensitive lipase), and LPL (lipoprotein lipase) are adipocyte-specific proteins expressed in differentiated adipocytes, serving as adipocyte differentiation markers capable of determining whether adipocytes have differentiated.

[0065] As shown in Figure 4, analysis of mRNA expression levels revealed that the mRNA expression levels of PPARγ, C / EBPα, adiponectin, HSL (Hormone-sensitive lipase), and LPL (lipoprotein lipase) were significantly increased in 3T3-L1 cells with completed adipogenesis (WC) compared to undifferentiated 3T3-L1 cells (NC). On the other hand, when SJF extract was applied at 4 μg / mL or 8 μg / mL during the adipogenesis process, the mRNA expression levels of PPARγ, C / EBPα, and adiponectin were found to decrease significantly in a concentration-dependent manner compared to 3T3-L1 cells with completed adipogenesis (WC).

[0066] In addition, as shown in Figure 5, analysis of protein expression levels revealed that the protein expression levels of PPARγ and C / EBPα were significantly increased in 3T3-L1 cells with completed adipogenesis (WC) compared to undifferentiated 3T3-L1 cells (NC). On the other hand, when 8 μg / mL of Siberian japonica extract (SJF) was applied during the adipogenesis process, the protein expression levels of PPARγ and C / EBPα were significantly decreased compared to 3T3-L1 cells with completed adipogenesis (WC).

[0067] The above results demonstrate that the extract of the Japanese zelkova tree has the effect of inhibiting the differentiation of adipocytes and intracellular lipid accumulation by suppressing the expression of transcription factors involved in the differentiation of adipocytes and intracellular lipid accumulation.

[0068] Foregoing, specific parts of the present invention have been described in detail. It is evident to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. That is, the actual scope of the invention is defined by the appended claims and their equivalents.

Claims

Claim 1 A pharmaceutical composition for the prevention or treatment of obesity comprising an extract of the Japanese spindle tree as an active ingredient, wherein the extract of the Japanese spindle tree inhibits the process of differentiation of preadipocytes into adipocytes and inhibits intracellular lipid accumulation. Claim 2 In paragraph 1, the above-mentioned Japanese zelkova extract is Japanese zelkova ( Sapium japonicum A pharmaceutical composition characterized by being a fruit extract of (Siebold & Zucc.) Pax & Hoffm. Claim 3 delete Claim 4 delete Claim 5 A pharmaceutical composition according to claim 1, characterized in that the extract of the Japanese zelkova inhibits the expression of PPARγ, C / EBPα, and adiponectin within cells. Claim 6 A health functional food composition for the prevention or improvement of obesity comprising a Japanese spindle tree extract as an active ingredient, wherein the Japanese spindle tree extract inhibits the process of differentiation of preadipocytes into adipocytes and inhibits intracellular lipid accumulation.

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